Maintaining a high concentration of K+ and a low concentration of Na+ in the cytosol is important for virtually all cells. The regulatory pathways controlling intracellular K+ and Na+ homeostasis are poorly understood in higher eukaryotic organisms. The proposed research will dissect the signaling cascades controlling K+ and Na+ homeostasis in the multicellular eukaryotic model organism Arabidopsis thaliana. Using a novel genetic screen, we have identified three genetic loci that are essential for K+ and Na+ homeostasis in Arabidopsis in response to Na+ stress. We have cloned one of the loci, SOS3 (for Salt Overly Sensitive 3), which encodes an EF-hand type calcium- binding protein with unique sequence features. We hypothesize that the SOS3 protein responds to a cytosolic calcium signal generated by Na+ stress and then regulates the activities of a protein kinase and/or phosphatase which in turn modulate the expression and/or activities of K+ and Na+ transporters. We propose to identify other components in the SOS3 pathway by using several cell biological and genetic approaches. In one approach, we will use the SOS3 protein as a bait to identify its interacting proteins using a yeast two-hybrid screen and through expression library screening with 32P-labeled SOS3. In another approach, we will identify genetic suppressors and enhancers of the sos3 mutation. Importantly, we will continue our current work to clone the SOS1 and SOS2 loci which have been implicated to function in the same pathway as SOS3. In addition, we plan to further analyze the expression and biochemical characteristics of the SOS3 protein to better understand its role as a calcium sensor for ion homeostasis regulation.